Process for producing hydroxycarboxylic acid

ABSTRACT

An object of the invention is to produce a microorganism which yields high amounts in a short time of a hydroxycarboxylic acid reduced in impurity content. This invention further provides a process for producing a hydroxycarboxylic acid, including glycolic acid, using the microorganism. This process enables a hydroxycarboxylic acid having high purity to be supplied at low cost.

TECHNICAL FIELD

The present invention relates to a microorganism which produces ahydroxycarboxylic acid including glycolic acid and a process forproducing the hydroxycarboxylic acid including glycolic acid using thesame.

BACKGROUND ART

Since some substances belonging to hydroxycarboxylic acids are useful asa raw material for polymers or an intermediate for medicines, a processfor effectively producing such substances have been in demand.

As an example, glycolic acid (α-hydroxyacetic acid) can be cited.Glycolic acid has been used as a cleaning agent or a raw material forcosmetics, but has recently been paid attention to as a raw material ofpolyglycolic acid which is useful as a gas barrier polymer or a medicalpolymer. The reason why glycolic acid has been paid attention to as agas barrier material is that the layer of polyglycolic acid has theproperty of a high oxygen barrier and is provided with performance as amaterial for packing food or carbonated beverage which goes easily badin the presence of oxygen.

In order to actually produce polyglycolic acids on the industrial scalein the future, glycolic acid that is its raw material must be suppliedin high purity and at low cost. However, glycolic acid of a chemicallysynthesized product which is currently available from the marketcontains not a few impurity contents, so there is a problem as a rawmaterial for polymers in terms of purity. It is because these impuritycontents not only prevent a dehydrative condensation reaction ofglycolic acid, but also methoxy acetate that is one of these impuritycontents is a compound suspicious of carcinogenic potential. Therefore,it is not desirable that such impurity contents are included in apacking material for food or beverage. Of course, it is technicallypossible to remove impurity contents by purification, but such purifiedproducts actually involve high cost. Thus, such purified products arenot realistic as a raw material for packing at low cost.

In order to avoid the aforementioned problem shown in glycolic acid ofthe chemically synthesized product, production of glycolic acid usingethylene glycol (hereinafter may be referred to as EG) as a raw materialhas been attempted according to the biological method. In JapanesePatent Laid-open Nos. H10-174593 (1998-174593) and H10-174594(1998-174594), there has been disclosed a method for producing glycolicacid by a microorganism, which comprises culturing yeast belonging toPichia (genus), Rhodotorula (genus), Sporobolomyces (genus),Kluyveromyces (genus) or Torulopsis (genus), a strain belonging toNocardia (genus), a strain belonging to Rhodococcus (genus), or anEscherichia coli B strain in a medium containing ethylene glycol forseparating and collecting glycolic acid from the culture broth.Incidentally, there has been disclosed that the yield of glycolic acidis low relative to Escherichia coli K12 strain. Of the methods forproducing glycolic acid as described in Examples of Japanese PatentLaid-open Nos. H10-174593 (1998-174593) and H10-174594 (1998-174594), amethod comprising employing Pichia naganishii results in the highestaccumulation concentration of glycolic acid, obtaining 35.3 g/L ofglycolic acid in a reaction for 30 hours. The production of glycolicacid employing Pichia naganishii has been reported in a literature(Kataoka, M., et al., Biosci. Biotechnol. Biochem., Vol. 65 (10) pp.2265-2270 (2001)) that 105 g/L of glycolic acid is obtained by areaction for 120 hours due to further improvement in the reactionconditions. In short, in a method for producing glycolic acid employingPichia naganishii, the time required for a reaction from ethylene glycolto glycolic acid is long, that is 120 hours, which causes an increase inthe production cost of glycolic acid. So, the reaction time has beenrequired to be reduced when the actual production on the industrialscale is supposed. And there has been a problem such that, in thisproduction method, by-product organic acids produced by a microorganismin use are entrained, thus causing difficulties in a purificationprocess or a dehydrative condensation reaction thereafter.

Regarding a metabolic reaction from ethylene glycol to glycolic acid bya microorganism, the results of study using Escherichia coli conductedby Boronat et al. have been disclosed (Boronat, A., et al., J.Bacteriol., Vol. 153 (1), pp. 134-139, (1983)), namely, a two-stagemetabolic pathway including a reaction from ethylene glycol toglycolaldehyde and a reaction from glycolaldehyde to glycolic acid.Boronat et al. have paid attention to the fact that propanedioloxidoreductase (hereinafter may be referred to as PDO redox enzyme) of acatalytic enzyme of a reaction from 1,2-propanediol (hereinafter may bereferred to as PDO) to lactaldehyde also recognizes ethylene glycol as asubstrate (Boronat, A., et al., Biochim. Biophys. Acta, Vol. 672, pp.98-107, (1981)). A strain which has the enhanced activity ofglycolaldehyde dehydrogenase (hereinafter may be referred to as GALdehydrogenase) of a catalytic enzyme of a reaction from glycolaldehydeto glycolic acid is isolated from strains in which activity of the PDOredox enzyme is enhanced by mutation. Incidentally, Table 1 below iscited from a thesis by Boronat et al.

TABLE 1 Activity of Activity of PDO redox GAL enzyme dehydrogenase PDOEG (U/mg (U/mg utilizing utilizing Strains protein) protein) capabilitycapability G1 strain 0.005 0.145 No No (referenced strain) G3 strain0.130 0.165 Yes No EG3 strain 0.415 0.560 (not Yes mentioned)

An Escherichia coli G1 strain of the referenced strain is able toutilize neither of 1,2-propanediol nor ethylene glycol. A G3 strain thatis a mutant strain in which activity of the PDO redox enzyme isremarkably enhanced has acquired the PDO utilizing capability, but stillcannot be able to utilize EG. On the basis of these results, Boronat etal. have reasoned that the reason why the G3 strain cannot be able toutilize EG might be that a reaction from glycolaldehyde to glycolic aciddoes not proceed due to insufficient activity of GAL dehydrogenase. Theyhave concluded that their reasoning is right because a mutant EG3 strainin which activity of GAL dehydrogenase is remarkably enhanced isisolated, and this strain utilizes EG. In other words, Boronat et al.insist that activity of GAL dehydrogenase must be sufficient in order toproduce glycolic acid from EG.

On the other hand, a new knowledge disclosed in the presentspecification is that enhancement of GAL dehydrogenase activity is notnecessarily required in order to produce glycolic acid from EG, but onlyenhancement of activity of PDO redox enzyme may be sufficient. Such adifference is caused because Boronat et al. have only paid attention toenhancement of GAL dehydrogenase activity in the EG3 strain and have notpaid attention to enhancement of activity of PDO redox enzyme as well atthe same time. Considering the test results of Boronat et al. againbased on our knowledge, the EG3 strain is able to acquire the EGutilizing capability not because activity of GAL dehydrogenase isenhanced, but because activity of PDO redox enzyme is enhanced.

Furthermore, according to a report by Boronat et al., in the EG3 strain,glycolaldehyde that is an intermediate is accumulated under thecondition in the presence of glycolic acid along with EG as a substrate.Namely, this shows to cause troubles such that glycolaldehyde that is anintermediate is accumulated as impurity content when glycolic acid isproduced from EG as a raw material according to the biological methodusing Escherichia coli.

According to the past knowledge from the aforementioned facts, it isshown that enhancement of activity of GAL dehydrogenase is absolutelynecessary when glycolic acid is produced from EG using Escherichia coliand glycolaldehyde that is an intermediate is accumulated in theproduced glycolic acid. From such a reason, even the skilled person inthe art wasn't able to easily suppose that glycolic acid can beselectively produced without enhancement of GAL dehydrogenase activityand glycolic acid can be even produced with good efficiency withoutincluding glycolaldehyde of an intermediate.

Various investigations of methods for producing glyceric acid as exampleof hydroxycarboxylic acids besides glycolic acid have also beenperformed. In such investigations, as a method for producing glycericacid using inexpensive glycerol as a raw material, a chemical synthesismethod comprising using a Pt catalyst has been disclosed in JapanesePatent Laid-open No. H5-331100 (1993-331100) and a biological method bya microorganism belonging to Gluconobacter (genus) has been disclosed inJapanese Patent Laid-open No. H1-168292 (1989-168292). In the formermethod comprising using a Pt catalyst, the reaction selectivity of about80% is never sufficient, thus generating by-products in a large amountand there is shown about a need to more strictly control the reactiontemperature. In the latter biological method, a method for producingD-glyceric acid using glycerine as a raw material has been described,but there is a problem in that it takes a long time of 4 days requiredfor the preparation of a microbial mass and the reaction and it ispossible to easily suppose that a large amount of by-product organicacids derived from a microorganism in use are entrained into theprepared glyceric acid. By the way, there has not been known about amethod for producing L-glyceric acid.

Furthermore, as an example of hydroxycarboxylic acid, a method forproducing hydroxyethoxy acetate according to the biological method hasbeen disclosed in Japanese Patent Laid-open No. S59-85296 (1984-85296).The method comprises culturing yeast belonging to Candida (genus),Torulopsis (genus), Rhodotorula (genus), Hansenula (genus), Debaryomyces(genus), Cryptococcus (genus) and Pichia (genus) in a medium containingdiethylene glycol, separating and collecting hydroxyethoxy acetate fromthe culture broth. In this production method, it is shown that it takesa long time required for culturing and diglycolic acid in a large amountis also produced as a by-product. From the fact, it is easily imaginedthat great efforts for the separation and purification of hydroxyethoxyacetate are needed.

-   Patent Document 1: Japanese Patent Laid-open No. H10-174593    (1998-174593)-   Patent Document 2: Japanese Patent Laid-open No. H10-174594    (1998-174594)-   Patent Document 3: Japanese Patent Laid-open No. H5-331100    (1993-331100)-   Patent Document 4: Japanese Patent Laid-open No. H1-168292    (1989-168292)-   Patent Document 5: Japanese Patent Laid-open No. S59-85296    (1984-85296)-   Non-patent Document 1: Kataoka, M., et al., Biosci. Biotechnol.    Biochem., Vol. 65 (10), pp. 2265-2270, (2001)-   Non-patent Document 2: Boronat, A., et al., J. Bacteriol., Vol. 153    (1), pp. 134-139, (1983)-   Non-patent Document 3: Boronat, A., et al., Biochim. Biophys. Acta,    Vol. 672, pp. 98-107, (1981)

DISCLOSURE OF THE INVENTION

As described above, various methods for producing a hydroxycarboxylicacid including glycolic acid in the past has drawbacks such that thehydroxycarboxylic acid obtained by the chemical synthesis method hasgenerally low purity because of impurity content contained therein in alarge amount, the productivity per unit hour of facilities becomes lowbecause of the long reaction time in the biological method, thusincreasing the production cost, and by-product organic acids areentrained. Under the above circumstances, an object of the presentinvention to solve is to supply hydroxycarboxylic acids includingglycolic acid in a short time at low cost and in large quantities, andto enhance the purity by reducing by-products.

As an improvement plan to solve the above object, the present inventorshave found that a process for producing glycolic acid from ethyleneglycol is suitable for the object of the present invention by using amicroorganism which has fully enhanced activity of the enzyme alonewhich is changed by a method comprising introducing a gene encoding anenzyme catalyzing a reaction from ethylene glycol to glycolaldehyde inthe form of plasmid or the like as a substitute of a microorganismselected from the natural system which has been used for the productionof glycolic acid according to the biological method in the past. And thepresent inventors have found a desired effect that cannot be expected tobe able to reduce the amount of glucose necessary for the culture of amicrobial mass by using a microorganism in which activity of an enzymecatalyzing a reaction from ethylene glycol to glycolaldehyde andactivity of an enzyme catalyzing a reaction from glycolaldehyde toglycolic acid are enhanced by introducing a gene encoding the twoenzymes in the form of plasmid as compared to a microorganism in whichonly activity of an enzyme catalyzing a reaction from ethylene glycol toglycolaldehyde is enhanced. In addition, the present inventors havefound that the aforementioned microorganism is capable of producingglycolic acid with much higher purity (small amount of by-productorganic acids) because by-product organic acids such as oxalic acid oracetic acid can be decreased by reducing activity of an enzymecatalyzing a reaction from glycolic acid to glyoxilic acid and/oractivity of an enzyme catalyzing a reaction from pyruvic acid to formicacid. The present inventors have also found that a situation in a cellbecomes more suitable for the production of glycolic acid by thereduction in activity of these two enzymes so that the productivity ofglycolic acid is considerably improved, though details are not clear.Furthermore, the present inventors have found these microorganisms canbe also used for a method for producing hydroxycarboxylic acids inaddition to glycolic acid.

That is, the present invention is specified by the following mattersfrom [1] to [9]:

[1] a process for producing hydroxycarboxylic acids with aliphaticpolyhydric alcohols having a hydroxyl group at the end as a substrate,which comprises using a microorganism which has the imparted or enhancedactivity of an enzyme catalyzing a reaction from ethylene glycol toglycolaldehyde;

[2] the production process as set forth in [1], which comprisesimparting or enhancing activity of the enzyme of the microorganism byintroducing a gene encoding the enzyme as set forth in [1] in the formof plasmid;

[3] a process for producing hydroxycarboxylic acids with aliphaticpolyhydric alcohols having a hydroxyl group at the end as a substrate,which comprises using a microorganism which has the imparted or enhancedactivity of both an enzyme catalyzing a reaction from ethylene glycol toglycolaldehyde and an enzyme catalyzing a reaction from glycolaldehydeto glycolic acid by introducing a gene encoding the two enzymes in theform of plasmid;

[4] the production process as set forth in [2] or [3], which comprisesusing a microorganism expressing the enzyme by functionally linking agene encoding an enzyme catalyzing a reaction from ethylene glycol toglycolaldehyde and/or a gene encoding an enzyme catalyzing a reactionfrom glycolaldehyde to glycolic acid to a promoter of the gene whichcontrols expression of a protein involved in a glycolytic pathway, anucleic acid biosynthesis pathway or an amino acid biosynthesis pathway;

[5] the production process as set forth in any one of [1] to [4], inwhich the enzyme catalyzing a reaction from ethylene glycol toglycolaldehyde is lactaldehyde reductase, and the enzyme catalyzing areaction from glycolaldehyde to glycolic acid is lactaldehydedehydrogenase;

[6] the production process as set forth in any one of [1] to [5], whichcomprises using a microorganism in which activity of glycolate oxidaseinherent in the microorganism is inactivated or decreased, and/oractivity of pyruvate formate-lyase is inactivated or decreased;

[7] the production process as set forth in any one of [1] to [6], inwhich the hydroxycarboxylic acid is a hydroxycarboxylic acid havingoptical activity;

[8] a microorganism used for the production process as set forth in anyone of [1] to [7]; and

[9] an aqueous solution of glycolic acid obtained by using ethyleneglycol as set forth in [6] as a substrate with the yield of not lessthan 95% and the production rate of not less than 2 g/L/hr.

Effect of the Invention

Since a microorganism which highly yields glycolic acid reduced impuritycontent in a much shorter time than the ones known up to now accordingto the present invention is produced, glycolic acid having high puritywhich can be used as a raw material for polymers can be industriallyproduced at low cost. Furthermore, by applying the aforementionedproduction process, hydroxycarboxylic acids can also be produced inaddition to glycolic acid.

Meanwhile, since glycolic acid can be highly produced with only onetarget enzyme in which its activity must be enhanced, which is contraryto the known information on the production of glycolic acid, effortsrequired for enhancement of activity of the enzymes can be reduced, andat the same time, undesired adverse effects on a host microorganism suchas dropout of the introduced gene due to stresses caused by artificiallyenhancing activity of a plurality of enzymes can be avoided.

Furthermore, by reducing glucose necessary for the culture of amicroorganism to be supplied for the production of hydroxycarboxylicacids, a method which reduces the production cost is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph which shows the time course of an amount of glycolicacid accumulated in the reaction solution in Example 3. In the figure,the triangle shows the results of Escherichia coli MG1655/pGAPfucOstrain, while the circle shows the results of Escherichia coli MG1655wild strain.

FIG. 2 is a graph which shows the time course of an amount of glycolicacid accumulated in the reaction solution in Example 4. In the figure,the square shows the results of Escherichia coli MG1655/pGAPfucO-aldAstrain, while the circle shows the results of Escherichia coli MG1655wild strain.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in more detail below.

In the present invention, the enzyme catalyzing a reaction from ethyleneglycol to glycolaldehyde is not particularly limited as far as it is anenzyme capable of producing glycolaldehyde using ethylene glycol as areaction substrate, and indicates all of such enzymes. As such anenzyme, lactaldehyde reductase can be cited.

In the present invention, the microorganism which has the impartedactivity of a certain target enzyme refers to a microorganism which hasnot activity of the enzyme at all, but which is provided with activityof the enzyme according to any method. In the present invention, themicroorganism which has the enhanced activity of a certain target enzymerefers to a microorganism in which activity of the enzyme isconsiderably enhanced as compared to the wild type microorganism. Thesemicroorganisms can be produced, for example, by using a method ofintroducing a gene encoding the enzyme into a wild type microorganism(or a microorganism before recombination) using a gene recombinationtechnique or the like. As a method for introducing the gene into thewild type microorganism (or a microorganism before recombination), amethod for introducing the gene into the microorganism in the form ofplasmid can be cited. Preparation of the genome DNA used forintroduction of a gene into a microorganism, preparation of plasmid,digestion and ligation of DNA, transformation, PCR (Polymerase ChainReaction), design and synthesis of oligonucleotide used as a primer andthe like can be carried out according to a usual method well known tothe skilled person in the art. These methods have been disclosed inSambrook, J., et al., “Molecular Cloning A Laboratory Manual, SecondEdition”, ColdSpring Harbor Laboratory Press, (1989) and the like.

The microorganism in the present invention refers to a generic name of amicroorganism capable of having a capability to produce ahydroxycarboxylic acid from aliphatic polyhydric alcohol having ahydroxyl group at the end by using any means regardless of whether itinherently has a capability to produce a hydroxycarboxylic acid fromaliphatic polyhydric alcohol having a hydroxyl group at the end. As sucha microorganism, Escherichia coli can be cited.

In the present invention, the aliphatic polyhydric alcohol having ahydroxyl group at the end has a hydroxyl group at the end of a carbonchain and is an aliphatic compound having two or more hydroxyl groups inthe molecule. In such a case, its structure is not particularly limited.Examples of such a compound include ethylene glycol, diethylene glycol,triethylene glycol, tetraethylene glycol, glycerol, 1,3-propanediol,1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1,2,4-butanetriol andthe like.

“In the form of plasmid” when introducing a gene encoding a certaintarget enzyme into a microorganism in the present invention refers topreparation of a recombinant plasmid by linking the gene to a vector andintroduction of the prepared plasmid into the microorganism by a methodof transformation or the like. Also, when the desired gene isfunctionally linked to a strong promoter constantly functioning in amicroorganism as described in Examples of the present invention, it ispossible to achieve the object of the present invention by using aplasmid in which the number of copies per microorganism cell isgenerally said to be low because of a property of replicon in a plasmid.As the plasmid having such a replicon, pACYC184 (GenBank accessionnumber: X06403) and the like can be cited.

The enzyme catalyzing a reaction from glycolaldehyde to glycolic acid inthe present invention is not particularly limited as far as it is anenzyme capable of producing glycolic acid using glycolaldehyde as areaction substrate, and indicates all such enzymes. As such an enzyme,lactaldehyde dehydrogenase can be cited.

In the present invention, the promoter of the gene which controlsexpression of the protein involved in the glycolytic pathway, thenucleic acid biosynthesis pathway or the amino acid biosynthesis pathwayrefers to a strong promoter which constantly functions in amicroorganism, and is less susceptible to suppression of expression evenin the presence of glucose. Specific examples thereof include a promoterof glyceraldehyde 3-phosphate dehydrogenase (hereinafter may be referredto as GAPDH) or a promoter of serine hydroxymethyl transferase.

The promoter in the present invention refers to a site, to which RNApolymerase having a sigma factor is bound and initiates transcription.For example, GAPDH promoter derived from an Escherichia coli is recordedin Base Nos. 397 to 440 in the base sequence information of GenBankaccession number X02662.

In the present invention, the gene functionally linked to the promoterrefers to a state that the gene is under control of promoter and thegene is bound to the promoter such that expression of the gene isproceeded by control of the promoter.

The lactaldehyde reductase in the present invention is classified intothe enzyme number 1.1.1.77, based on the report of the enzyme committeeof International Union of Biochemistry (I.U.B.), and refers to a genericname of an enzyme which reversibly catalyzes a reaction to producelactaldehyde from 1,2-propanediol in the presence of oxidizednicotinamide adenine dinucleotide which is a coenzyme.

The lactaldehyde dehydrogenase in the present invention is classifiedinto the enzyme number 1.2.1.22, based on the report of the enzymecommittee of International Union of Biochemistry (I.U.B.), and refers toa generic name of an enzyme that catalyzes a reaction to produce lacticacid from lactaldehyde in the presence of oxidized nicotinamide adeninedinucleotide which is a coenzyme. It is also classified into the enzymenumber 1.2.1.21, based on the report of the enzyme committee ofInternational Union of Biochemistry (I.U.B.), and refers to a genericname of an enzyme glycolaldehyde dehydrogenase that catalyzes a reactionto produce glycolic acid from glycolaldehyde in the presence of oxidizednicotinamide adenine dinucleotide which is a coenzyme. This is because,in the prior literature using Escherichia coli, there has been reportedthat lactaldehyde dehydrogenase and glycolaldehyde dehydrogenase are thesame enzyme (Caballero, E., et al., J. Biol. Chem., Vol. 258 (12), pp.7788-7792 (1983)).

Gglycolate oxidase (hereinafter may be referred to as glcDEF) in thepresent invention is classified into the enzyme number 1.1.3.15, basedon the report of the enzyme committee of International Union ofBiochemistry (I.U.B.), and refers to a generic name of an enzyme whichreversibly catalyzes a reaction to produce glyoxilic acid from glycolicacid.

Pyruvate formate-lyase (hereinafter may be referred to as pflB) in thepresent invention is classified into the enzyme number 2.3.1.54, basedon the report of the enzyme committee of International Union ofBiochemistry (I.U.B.), and refers to a generic name of an enzyme whichreversibly catalyzes a reaction to produce formic acid from pyruvicacid.

Inactivation of functions of an enzyme glcDEF or pflB in the presentinvention means complete loss of activity of the enzyme. Decrease infunctions of an enzyme glcDEF or pflB in the present invention meanspartial loss of activity of the enzyme. In order to inactivate ordecrease functions of the enzyme, there are methods such as introducinga mutation into the gene encoding the protein, eliminating the gene,adding a drug which specifically inactivates the enzyme, irradiating theenzyme with ultraviolet rays or the like. Specifically, an Escherichiacoli MT-11023 strain can be cited as a microorganism in which functionsof these enzymes are inactivated by disruption of glcDEF gene and pflBgene.

Since the Escherichia coli MT-11023 strain is a strain in which glcDEFand pflB are inactivated by gene disruption, the present invention canbe carried out easily using this strain. The present strain has beendeposited on Mar. 10, 2005 as the deposition number FERM BP-10293 atInternational Patent Organism Depository Center, Tsukuba Central 6,1-1-1 Higashi, Tsukuba, Ibaraki, of National Institute of AdvancedIndustrial Science and Technology, based on the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure.

When carrying out the production process of the present invention, amicroorganism microbial mass in a necessary amount is obtained usuallyby culturing and growing a microorganism which has the imparted orenhanced activity of an enzyme catalyzing a reaction from ethyleneglycol to glycolaldehyde using a medium.

The medium to be used for the culture according to the present inventionis not particularly limited if it is a medium containing carbon source,nitrogen source, inorganic ion and as needed traces of other organiccomponents. As carbon source, saccharides such as glucose, fructose,molasses and the like; organic acids such as fumaric acid, citric acid,succinic acid and the like; and alcohols such as methanol, ethanol,glycerol and the like are properly used. As nitrogen source, inorganicnitrogen sources such as organic ammonium salts, inorganic ammoniumsalts, ammonia gas, ammonia water and the like; and organic nitrogensources such as protein hydrolysates and the like are properly used. Asinorganic ion, magnesium ion, phosphate ion, potassium ion, iron ion,manganese ion and others are properly used as required. As traces oforganic components, vitamin, amino acid or the like and yeast extractscontaining vitamin, amino acid or the like, peptone, corn steep liquor,casein hydrolysate and the like are properly used.

Incidentally, as the medium to be used for the culture according to thepresent invention, preferably used is a liquid medium considering that amicroorganism is provided for the industrial production.

When the microorganism is cultured according to the present invention,the culture condition is not particularly limited. For example, when themicroorganism is cultured while properly controlling pH and temperaturein a pH range of 6 to 8, a temperature range of 25 to 40 degreecentigrade under aerobic conditions, the time required for the cultureis within 50 hours.

The reaction solution to be used for a reaction to producehydroxycarboxylic acids according to the present invention is notparticularly limited if it is a solution capable of adding (orcontaining) aliphatic polyhydric alcohol having a hydroxyl group at theend to be a substrate. Examples thereof include a buffer solution suchas a potassium phosphate buffer solution and the like or a medium usedfor the culture of the aforementioned microorganism and pure water. Inthe present invention, a reaction is carried out by bringing themicroorganism microbial mass previously obtained by the culture intocontact with this solution. The microorganism microbial mass in use canbe the culture broth itself wherein the culture is completed, or can bea microbial mass alone recovered from the culture broth, prior to use.

Upon the reaction in a process for producing hydroxycarboxylic acids ofthe present invention, the reaction condition is not particularlylimited. But, for example, the reaction is preferably carried out whileappropriately controlling pH and temperature in a pH range of 6 to 9, atemperature range of 20 to 40 degree centigrade.

A process for recovering a hydroxycarboxylic acid accumulated in thereaction solution obtained as described above is not particularlylimited. But, there can be adopted, for example, a process comprisingremoving the microbial mass from the reaction solution by centrifugationor the like and then using a synthetic adsorbent resin, a process usinga precipitant, a process comprising separating a hydroxycarboxylic acidaccording to other usual collection and separation methods.

EXAMPLES Example 1 Construction of Escherichia coli-Derived LactaldehydeReductase Expression Vector and a Transformant of the Expression Vector

The amino acid sequence and the base sequence of a gene (hereinafter maybe simply referred to as fucO) of lactaldehyde reductase of Escherichiacoli have been already reported (GenBank accession number: M31059). Inorder to acquire fucO, CGAATTCCGGAGAAAGTCTTATGATGGCTAACAGAATGATTCTG(Sequence No. 1) and GTGAAGCTTGCATTTACCAGGCGGTATGG (Sequence No. 2) wereused for a PCR amplification using the genome DNA of Escherichia coliMG1655 strain as a template, and the obtained DNA fragment was digestedwith restriction enzymes EcoRI and HindIII to give a fucO fragment ofabout 1.2 kbp. Furthermore, in order to acquire a glyceraldehyde3-phosphate dehydrogenase (GAPDH) promoter,AACGAATTCTCGCAATGATTGACACGATTC (Sequence No. 3) andACAGAATTCGCTATTTGTTAGTGAATAAAAGG (Sequence No. 4) were used for a PCRamplification using the genome DNA of Escherichia coli MG1655 strain asa template, and the obtained DNA fragment was digested with arestriction enzyme EcoRI to give a DNA fragment of about 100 bp whichencodes a GAPDH promoter. The above-mentioned two DNA fragments weremixed with the fragment obtained by digestion of plasmid pUC18 withrestriction enzymes EcoRI and HindIII, ligated using a ligase, and thentransformed to an Escherichia coli DH5 strain, to give a transformantwhich grows on an LB agar plate containing 50 μg/mL of ampicillin. Theobtained colony was cultured in an LB liquid medium containing 50 μg/mLof ampicillin at 37 degree centigrade overnight, and a plasmid pGAPfucOwas recovered from the obtained microbial mass. This plasmid pGAPfucOwas transformed to an Escherichia coli MG1655 strain, and cultured on anLB agar plate containing 50 μg/mL of ampicillin at 37 degree centigradeovernight to give a MG1655/pGAPfucO strain.

Incidentally, the Escherichia coli MG1655 strain and Escherichia coliDH5 strain can be obtained from American Type Culture Collection.

Example 2 Construction of Lactaldehyde Reductase and LactaldehydeDehydrogenase Double-Expression Vector and a Transformant of theExpression Vector

The amino acid sequence and the base sequence of a gene (hereinafter maybe simply referred to as fucO) of lactaldehyde reductase of Escherichiacoli have been already reported (GenBank accession number: M31059).Furthermore, the amino acid sequence and the base sequence of a gene(hereinafter may be simply referred to as aldA) of lactaldehydedehydrogenase of Escherichia coli have been already reported as well(GenBank accession number: M64541). In order to acquire fucO,GCTCTAGACGGAGAAAGTCTTATGATGGCTAACAGAATGATTCTG (Sequence No. 5) andGTGAAGCTTGCATTTACCAGGCGGTATGG (Sequence No. 2) were used for a PCRamplification using the genome DNA of Escherichia coli MG1655 strain asa template, and the obtained DNA fragment was digested with restrictionenzymes XbaI and HindIII to give a fucO fragment of about 1.2 kbp.Furthermore, in order to acquire aldA,CGAATTCCGGAGAAAGTCTTATGTCAGTACCCGTTCAACATCC (Sequence No. 6) andGCTCTAGACTCTTTCACTCATTAAGACTG (Sequence No. 7) were used for a PCRamplification using the genome DNA of Escherichia coli MG1655 strain asa template, and the obtained DNA fragment was digested with restrictionenzymes EcoRI and XbaI to give an aldA fragment of about 1.5 kbp.Furthermore, a fragment encoding a glyceraldehyde 3-phosphatedehydrogenase (GAPDH) promoter was obtained in the same manner as inExample 1. The above-mentioned three DNA fragments were mixed with thefragment obtained by digestion of plasmid pUC18 with restriction enzymesEcoRI and HindIII, ligated using a ligase, and then transformed to anEscherichia coli DH5 strain, to give a transformant which grows on an LBagar plate containing 50 μg/mL of ampicillin. The obtained colony wascultured in an LB liquid medium containing 50 μg/mL of ampicillin at 37degree centigrade overnight, and a plasmid pGAPfucO-aldA was recoveredfrom the obtained microbial mass. This plasmid pGAPfucO-aldA wastransformed to an Escherichia coli MG1655 strain, and cultured on an LBagar plate containing 50 μg/mL of ampicillin at 37 degree centigradeovernight, to give a MG1655/pGAPfucO-aldA strain.

Example 3 Production of Glycolic Acid by Escherichia coliMG1655/pGAPfucO Strain and Escherichia coli MG1655 Wild Strain

The Escherichia coli MG1655/pGAPfucO strain obtained in Example 1 andEscherichia coli MG1655 wild strain were inoculated into 25 mL of LBBroth, Miller's culture solution (Difco244620) contained in a conicalflask, and the culture was carried out with stirring overnight with 120rpm at a culture temperature of 35 degree centigrade as preculture.Then, the whole amount of the respective preculture solutions wastransferred to a 1 L-fermentor (BMJ-01, culture apparatus manufacturedby ABLE Corporation) containing 475 g of the medium of the compositionshown in Table 2 to carry out culture. The culture was carried out underthe conditions including atmospheric pressure, an aeration rate of 1vvm, a stirring speed of 800 rpm, a culture temperature of 35 degreecentigrade and pH 7.2 (adjusted with an aqueous NH₃ solution). Themicrobial mass at 24 hours after starting the culture was collected bycentrifugation (8,000 rpm for 20 minutes) and washed with pH 7.2 of 1 mMpotassium phosphate buffer solution, and then suspended with this buffersolution to have 500 ml of final liquid amount. The suspension wastransferred to a fermentor of a culture apparatus BMJ-01 manufactured byABLE Corporation and ethylene glycol was added thereto at a rate of 5g/hr to carry out the reaction for 22 hours. The reaction was carriedout under the conditions including atmospheric pressure, an aerationrate of 1 vvm, a stirring speed of 800 rpm, a culture temperature of 35degree centigrade and pH 7.2 (adjusted with an aqueous NH₃ solution).The amount of glycolic acid accumulated in the obtained reactionsolution was measured by HPLC according to an established method. Theresults are shown in FIG. 1.

It was confirmed that 106 g/L of glycolic acid was accumulated in theEscherichia coli MG1655/pGAPfucO strain at 22 hours, whileglycolaldehyde that is an intermediate from ethylene glycol to glycolicacid was not detected. On the other hand, in the wild strain, glycolicacid was not detected even after 22 hours of the reaction.

TABLE 2 Medium Composition Polypepton 7 g/L Glucose 30 g/L Fe₂SO₄ 0.09g/L K₂HPO₄ 2 g/L KH₂PO₄ 2 g/L MgSO₄•7H₂O 2 g/L (NH₄)₂SO₄ 1.5 g/L(Residue: Water)

Example 4 Production of Glycolic Acid by Escherichia coliMG1655/pGAPfucO-aldA Strain and Escherichia coli MG1655 Wild Strain

The Escherichia coli MG1655/pGAPfucO-aldA strain obtained in Example 2and Escherichia coli MG1655 wild strain were cultured in the same manneras in Example 3. After 24 hours from starting the culture, ethyleneglycol was directly added to the fermentor in culture at a rate of 5g/hr for 22 hours to carry out the reaction. The amount of glycolic acidaccumulated in the obtained culture broth was measured by HPLC accordingto an established method. The results are shown in FIG. 2.

It was confirmed that, in the Escherichia coli MG1655/pGAPfucO-aldAstrain, 110 g/L of glycolic acid was accumulated at 22 hours afterstarting the addition of ethylene glycol, while glycolaldehyde that isan intermediate from ethylene glycol to glycolic acid was not detected.On the other hand, in the wild strain, glycolic acid was not detectedeven at 22 hours after initiating the addition of ethylene glycol.

Example 5 Preparation of Escherichia coli MG1655aldA-Deleted Strain

The entire base sequence of the genome DNA of Escherichia coli is known(GenBank accession number: U00096), and the amino acid sequence and thebase sequence of a gene (hereinafter may be simply referred to as aldA)of lactaldehyde dehydrogenase of Escherichia coli have also beenreported (GenBank accession number: M64541). PCR was performed using theprimer pair of TACTGCAGTGATCCTTGCAGGCAATGC (Sequence No. 8) andGGTCTAGAATCATCAGAGAGACGGAATCG (Sequence No. 9), or the pair ofGGTCTAGAATGAGTGAAAGAGGCGGAG (Sequence No. 10) andAAGGTACCGATGCTGGTGCGAAGAAGG (Sequence No. 11), which were prepared onthe basis of the gene information of aldA-adjacent region of the genomeDNA of Escherichia coli MG1655 strain. Each of the obtained DNAfragments was digested with restriction enzymes PstI and XbaI, and XbaIand Kpnl, to give fragments of about 800 bp and 700 bp respectively.These DNA fragments were mixed with the fragment obtained by digestionof a temperature-sensitive cloning vector pTH18cs1 (GenBank accessionnumber: AB019610) [Hashimoto-Gotoh, T., Gene, Vol. 241, pp. 185-191(2000)] with PstI and KpnI, ligated using a ligase, and then transformedto a DH5 strain at 30 degree centigrade, to give a transformant whichgrows on an LB agar plate containing 10 μg/ml of chloramphenicol. Theobtained colony was cultured in an LB liquid medium containing 10 μg/mlof chloramphenicol at 30 degree centigrade overnight, and a plasmid wasrecovered from the obtained microbial mass.

This plasmid was transformed to an Escherichia coli MG1655 strain at 30degree centigrade, and cultured on an LB agar plate containing 10 μg/mlof chloramphenicol at 30 degree centigrade overnight to give atransformant. The obtained transformant was applied onto an LB liquidmedium containing 10 μg/ml of chloramphenicol and cultured at 30 degreecentigrade overnight. Next, in order to obtain the cultured microbialmass, the cultured microbial mass was applied onto an LB agar platecontaining 10 μg/ml of chloramphenicol to give colonies which grow at 42degree centigrade. The obtained colonies were cultured in an LB liquidmedium without containing an antibiotic at 30 degree centigradeovernight and further applied onto an LB agar plate without containingan antibiotic, to give colonies which grow at 42 degree centigrade.

From the grown colonies, 100 colonies were picked up randomly, and eachof them was grown on an LB agar plate without containing an antibioticand an LB agar plate containing 10 μg/ml of chloramphenicol, to selectchloramphenicol-sensitive clones which grow only on an LB agar platewithout containing an antibiotic. Furthermore, a fragment of about 3.2kb containing aldA was amplified by PCR using the chromosome DNA ofthese desired clones to select a strain in which aldA was deleted, andthe obtained strain was named as a MG1655aldA-deleted strain(hereinafter may be simply referred to as ΔaldA).

Example 6 Construction of ΔaldA/pGAPfucO Strain

The plasmid pGAPfucO obtained in Example 1 was transformed to the ΔaldAstrain obtained in Example 5 and cultured on an LB agar plate containing50 μg/mL of ampicillin at 37 degree centigrade overnight, to give aΔaldA/pGAPfucO strain.

Example 7 Production of Glycolic Acid by ΔaldA/pGAPfucO Strain

Each of ΔaldA/pGAPfucO strain and MG1655/pGAPfucO strain was cultured,collected and washed in the same manner as in Example 3, and thenethylene glycol was added to the obtained microbial mass in the samemanner as in Example 3 to carry out the reaction for 10 hours. It wasconfirmed that 64.8 g of glycolic acid was accumulated in theΔaldA/pGAPfucO strain and 64.2 g of glycolic acid was accumulated in theMG1655/pGAPfucO strain by the reaction. It was shown that lactaldehydedehydrogenase was not necessarily required for the production ofglycolic acid.

Example 8 Comparison of Microbial Mass Growth of Escherichia coliMG1655/pGAPfucO-aldA Strain and Escherichia coli MG1655/pGAPfucO Strain

The Escherichia coli MG1655/pGAPfucO-aldA strain obtained in Example 2and the Escherichia coli MG1655/pGAPfucO strain obtained in Example 1were cultured in the same manner as in Example 3. However, the culturewas carried out with the glucose amount of 70 g/L in the mediumcomposition shown in Table 1. After culturing for 24 hours, the entireculture broth was collected by centrifugation to measure the weight ofwet microbial mass obtained. Furthermore, the amount of residual glucosein the medium was measured according to an established method and theamount of glucose necessary for growth of the microbial mass wascalculated. The results are shown in Table 3.

Even though the weights of microbial mass obtained from these twostrains were almost the same, it was shown that the amount of glucosewhich was needed at that time could be 20% or more less in theMG1655/pGAPfucO-aldA strain. It was shown that lactaldehydedehydrogenase was not necessarily needed in the production of glycolicacid in Example 7, but it was confirmed that lactaldehyde dehydrogenasewas effective in the reduction of the amount of glucose necessary forthe production of glycolic acid due to the enhanced activity of theenzyme.

TABLE 3 MG1655/pGAPfucO MG1655/pGAPfucO-aldA strain strain Weight of wet45.3 45.5 microbial mass (g) Consumption of 31.6 24.5 glucose (g)

Example 9 Substitution of the fucO Promoter on the Genome of Escherichiacoli MG1655 Strain with GAPDH Promoter

The entire base sequence of the genome DNA of Escherichia coli is known(GenBank accession number: U00096), and the base sequence of fucO ofEscherichia coli has also been reported (GenBank accession number:M31059). PCR was performed using GCTCTAGACCTTCTCCTTGTTGCTTTACG (SequenceNo. 12) and AACTGCAGAGAGAGGTAGCTAATGGAACG (Sequence No. 13), which wereprepared on the basis of the gene information of 5′-adjacent region offucO of the Escherichia coli MG1655 strain, using the genome DNA ofEscherichia coli as a template, to amplify a DNA fragment of about 650bp.

Furthermore, PCR was performed using GGTCTAGAGCAATGATTGACACGATTCG(Sequence No. 14) which was prepared on the basis of the sequenceinformation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoterof the Escherichia coli MG1655 strain, and CAGGTACCATCCTTATCACCAGCAACC(Sequence No. 15) which was prepared on the basis of the sequenceinformation of fucO of the Escherichia coli MG1655 strain, using theexpression vector pGAPfucO prepared in Example 1 as a template, to givea DNA fragment of about 730 bp which contains a GAPDH promoter and aninitiation codon-adjacent region of fucO.

Each of the fragments obtained above was digested with restrictionenzymes PstI and XbaI, and XbaI and KpnI, and this fragment was mixedwith the fragment obtained by digestion of temperature-sensitive plasmidpTH18cs1 (GenBank accession number: AB019610) [Hashimoto-Gotoh, T.,Gene, Vol. 241, pp. 185-191 (2000)] with PstI and KpnI, ligated using aligase, and then transformed to a DH5 strain at 30 degree centigrade, togive a transformant which grows on an LB agar plate containing 10 μg/mlof chloramphenicol. The obtained colony was cultured in an LB liquidmedium containing 10 μg/ml of chloramphenicol at 30 degree centigradeovernight, and a plasmid was recovered from the obtained microbial mass.This plasmid was transformed to a MG1655 strain at 30 degree centigrade,and cultured on an LB agar plate containing 10 μg/ml of chloramphenicolat 30 degree centigrade overnight to give a transformant. The obtainedtransformant was applied onto an LB liquid medium containing 10 μg/ml ofchloramphenicol and cultured at 30 degree centigrade overnight. Next, inorder to obtain the cultured microbial mass, the cultured microbial masswas applied onto an LB agar plate containing 10 μg/ml of chloramphenicolto give colonies which grow at 42 degree centigrade. The obtainedcolonies were cultured in an LB liquid medium without containing anantibiotic at 30 degree centigrade overnight and further applied onto anLB agar plate without containing an antibiotic to give colonies whichgrow at 42 degree centigrade.

From the grown colonies, 100 colonies were picked up randomly, and eachof them was grown on an LB agar plate without containing an antibioticand an LB agar plate containing 10 μg/ml of chloramphenicol, to selectchloramphenicol-sensitive clones. Furthermore, a fragment of about 730bp containing a GAPDH promoter and a fucO partial sequence was amplifiedby PCR using the chromosome DNA of these desired clones, to select astrain in which the fucO promoter region is substituted with the GAPDHpromoter, and the clone which satisfies the above description was namedas an MG1655fucO-deleted GAPpfucO genome-inserted strain.

Example 10 Production of Glycolic Acid by MG1655fucO-Deleted GAPpfucOGenome-Inserted Strain

The Escherichia coli MG1655fucO-deleted GAPpfucO genome-inserted strainwas cultured, collected and washed in the same manner as in Example 3,and then ethylene glycol was added to the obtained microbial mass in thesame manner as in Example 3 to carry out the reaction for 24 hours.Accumulation of glycolic acid was not confirmed.

Example 11 Construction of fucO Expression Vector Using a Low CopyVector

PCR was performed using AACGAATTCTCGCAATGATTGACACGATTC (Sequence No. 3)which was prepared on the basis of the sequence information of theglyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter of theEscherichia coli MG1655 strain and GTGAAGCTTGCATTTACCAGGCGGTATGG(Sequence No. 2) which was prepared on the basis of the sequenceinformation of fucO of the Escherichia coli MG1655 strain, using theexpression vector pGAPfucO prepared in Example 1 as a template, to givea DNA fragment of about 1,300 bp which contains a GAPDH promoter andfucO.

The DNA fragment was mixed with the fragment obtained by digestion ofplasmid pACYC184 (GenBank accession number: X06403) with a restrictionenzyme HincII, ligated using a ligase, and then transformed to anEscherichia coli DH5 strain, to give a transformant which grows on an LBliquid containing 20 μg/mL of chloramphenicol. The obtained colony wascultured in an LB liquid medium containing 20 μg/mL of chloramphenicolat 37 degree centigrade overnight, and a plasmid pACYCfucO was recoveredfrom the obtained microbial mass. This plasmid pACYCfucO was transformedto an Escherichia coli MG1655 strain, and cultured in an LB agar platecontaining 20 μg/mL of chloramphenicol at 37 degree centigrade overnightto give a MG1655/pACYCfucO strain.

Example 12 Production of Glycolic Acid by MG1655/pACYCfucO Strain

An Escherichia coli MG1655/pACYCfucO strain was cultured, collected andwashed in the same manner as in Example 3, and then ethylene glycol wasadded to the obtained microbial mass in the same manner as in Example 3to carry out the reaction for 9 hours. 28 g of glycolic acid wasaccumulated by the reaction.

Example 13 Measurement of Activity of Lactaldehyde Reductase of MG1655Wild Strain, MG1655fucO-deleted GAPpfucO genome-inserted strain,MG1655/pACYCfucO Strain and MG1655/pGAPfucO Strain

Each of microbial masses was cultured in the same manner as in Example3. The microbial mass after the culture was collected and washed, andthen suspended with pH 7.4 of 50 mM potassium phosphate buffer solutionand disrupted with ultrasonic waves. The microbial mass extract afterdisruption was centrifuged and the supernatant was used for themeasurement of activity of lactaldehyde reductase. Ethylene glycol wasused as a substrate and the increase in reduced nicotinamide adeninedinucleotide produced by the reaction was measured using aspectrophotometer to measure the activity. The amount of reducednicotinamide adenine dinucleotide generated for a minute per weight of asoluble protein used for the reaction was calculated, and the resultsare shown in Table 4 when the value of the MG1655 wild strain was 1.

In the MG1655fucO-deleted GAPpfucO genome-inserted strain, activity oflactaldehyde reductase was considerably increased to 18 times of theMG1655 wild strain. However, it was shown that such an increase inactivity was not sufficient for the production of glycolic acid, and theincrease in activity of at least about 1,700 times was needed asindicated by the case where the activity was enhanced by theintroduction of a low copy vector for the production of glycolic aciddue to the sole enhancement of lactaldehyde reductase.

TABLE 4 MG1655fucO-de- MG1655 leted GAPpfucO MG1655/ MG1655/ wildgenome-inserted pACYCfucO pGAPfucO strain strain strain strain Relative1 18.3 1783.7 3724.1 activity of lactaldehyde reductase

Example 14 Production of L-Glyceric Acid by Escherichia coliMG1655/pGAPfucO Strain and Escherichia coli MG1655 Wild Strain

Escherichia coli MG1655/pGAPfucO strain and Escherichia coli MG1655 wildstrain were cultured, collected and washed in the same manner as inExample 3, and then glycerol was added instead of ethylene glycol addedin Example 3 to the obtained microbial mass in a lump so as to have itsconcentration of 150 g/L to carry out the reaction for 24 hours with 500ml of final liquid amount. The amount of L-glyceric acid accumulated inthe obtained culture broth was measured by HPLC using a column forresolving optical isomers according to an established method. In theEscherichia coli MG1655/pGAPfucO strain, accumulation of 67 g/L ofL-glyceric acid was confirmed at 24 hours. In the Escherichia coliMG1655 wild strain, accumulation of L-glyceric acid was not confirmed.

Example 15 Production of Hydroxyethoxy Acetate by Escherichia coliMG1655/pGAPfucO Strain and Escherichia coli MG1655 Wild Strain

Escherichia coli MG1655/pGAPfucO strain and Escherichia coli MG1655 wildstrain were cultured in the same manner as in Example 3. The microbialmass at 24 hours after starting the culture was collected bycentrifugation (8,000 rpm for 20 minutes) and washed with pH 7.2 of 1 mMpotassium phosphate buffer solution. About 300 mg of the microbial massafter washing was suspended with the same buffer solution, and thereaction was carried out for 18 hours with 4 ml of final liquid amountin the presence of diethylene glycol in a final concentration of 100 mM.The reaction was carried out while stirring using a magnetic stirrerunder atmospheric pressure at a reaction temperature of 37 degreecentigrade. Hydroxyethoxy acetate in the obtained reaction solution wasmeasured with HPLC according to an established method. In theEscherichia coli MG1655/pGAPfucO strain, accumulation of 1 g/L ofhydroxyethoxy acetate was confirmed. Diethylene glycol-derivedby-product including diglycolic acid was not detected. Furthermore, inthe Escherichia coli MG1655 wild strain, accumulation of hydroxyethoxyacetate was not confirmed.

Example 16 Preparation of Escherichia coli MG1655glcDEF Gene-DeletedStrain

The entire base sequence of the genome DNA of Escherichia coli is known(GenBank accession number: U00096), and the base sequence of the glcDEFgene of Escherichia coli has been already reported (GenBank accessionnumber: L43490). PCR was performed using the primer pair ofTTGGTACCGTTCTGCCAGCAACTGACG (Sequence No. 16) andTGTCTAGAGTACCTCTGTGCGTCACTGG (Sequence No. 17), or the pair ofGCTCTAGACGCTTTGTTGTGTTGTGTGG (Sequence No. 18) andAACTGCAGGATCGGTCAATGATTGCAGC (Sequence No. 19), which were prepared onthe basis of the gene information of glcDEF gene-adjacent region of thegenome DNA of Escherichia coli MG1655. Each of the obtained DNAfragments was digested with restriction enzymes KpnI and XbaI, and XbaIand PstI, to give fragments of about 670 bp and 790 bp respectively.These DNA fragments were mixed with the fragment obtained by digestionof a temperature-sensitive cloning vector pTH18cs1 (GenBank accessionnumber: AB019610) [Hashimoto-Gotoh, T., Gene, Vol. 241, pp. 185-191(2000)] with KpnI and PstI, ligated using a ligase, and then transformedto a DH5 strain at 30 degree centigrade, to give a transformant whichgrows on an LB agar plate containing 10 μg/ml of chloramphenicol. Theobtained colony was cultured in an LB liquid medium containing 10 μg/mlof chloramphenicol at 30 degree centigrade overnight, and a plasmid wasrecovered from the obtained microbial mass. This plasmid was named aspTHΔglcDEF.

This plasmid pTHΔglcDEF was transformed to an Escherichia coli MG1655strain at 30 degree centigrade, and cultured on an LB agar platecontaining 10 μg/ml of chloramphenicol at 30 degree centigrade overnightto give a transformant. The obtained transformant was applied onto an LBliquid medium containing 10 μg/ml of chloramphenicol, and cultured at 30degree centigrade overnight. Next, in order to obtain the culturedmicrobial mass, the cultured microbial mass was applied onto an LB agarplate containing 10 μg/ml of chloramphenicol to give colonies which growat 42 degree centigrade. The obtained colonies were cultured in an LBliquid medium without containing an antibiotic at 30 degree centigradeovernight, and further applied onto an LB agar plate without containingan antibiotic to give colonies which grow at 42 degree centigrade.

From the grown colonies, 100 colonies were picked up randomly, and eachof them was grown on an LB agar plate without containing an antibioticand an LB agar plate containing 10 μg/ml of chloramphenicol, to selectchloramphenicol-sensitive clones which grow only on an LB agar platewithout containing an antibiotic. Furthermore, a fragment of about 3.8kb containing a glcDEF gene was amplified by PCR using the chromosomeDNA of these desired clones, to select a strain in which the glcDEF generegion was deleted, and the obtained strain was named as an MG1655glcDEFgene-deleted strain (hereinafter may be simply referred to as a ΔglcDEFstrain).

Example 17 Preparation of Escherichia coli MG1655pflB Gene-DeletedStrain

The entire base sequence of the pflB gene of Escherichia coli has beenalready reported (GenBank accession number: X08035). PCR was performedusing the primer pair of GCACGAAAGCTTTGATTACG (Sequence No. 20) andTTATTGCATGCTTAGATTTGACTGAAATCG (Sequence No. 21), or the pair ofTTATTGCATGCTTATTTACTGCGTACTTCG (Sequence No. 22) andAAGGCCTACGAAAAGCTGCAG (Sequence No. 23), which were prepared on thebasis of the gene information of pflB gene-adjacent region of thechromosome DNA of MG1655 strain. Each of the obtained DNA fragments wasdigested with restriction enzymes HindIII and SphI, and SphI and PstI,to give fragments of about 1,770 bp and 1,340 bp respectively. These DNAfragments were mixed with the fragment obtained by digestion of atemperature-sensitive cloning vector pTH18cs1 (GenBank accession number:AB019610) [Hashimoto-Gotoh, T., Gene, Vol. 241, pp. 185-191 (2000)] withHindIII and PstI, ligated using a ligase, and then transformed to a DH5strain at 30 degree centigrade, to give a transformant which grows on anLB agar plate containing 10 μg/ml of chloramphenicol. The obtainedcolony was cultured in an LB liquid medium containing 10 μg/ml ofchloramphenicol at 30 degree centigrade overnight, and a plasmid wasrecovered from the obtained microbial mass. This plasmid was transformedto an Escherichia coli MG1655 strain at 30 degree centigrade, andcultured on an LB agar plate containing 10 μg/ml of chloramphenicol at30 degree centigrade overnight to give a transformant. The obtainedtransformant was applied onto an LB liquid medium containing 10 μg/ml ofchloramphenicol and cultured at 30 degree centigrade overnight. Next, inorder to obtain the cultured microbial mass, the cultured microbial masswas applied onto an LB agar plate containing 10 μg/ml of chloramphenicolto give colonies which grow at 42 degree centigrade. The obtainedcolonies were cultured in an LB liquid medium without containing anantibiotic at 30 degree centigrade overnight and further applied onto anLB agar plate without containing an antibiotic to give colonies whichgrow at 42 degree centigrade.

From the grown colonies, 100 colonies were picked up randomly, and eachof them was grown on an LB agar plate without containing a drug and anLB agar plate containing 10 μg/ml of chloramphenicol, to selectchloramphenicol-sensitive clones which grow only on an LB agar platewithout containing an antibiotic. Furthermore, a fragment of about 2.0kb containing a pflB gene was amplified by PCR using the chromosome DNAof these desired clones, to select a strain in which the pflB generegion was deleted, and the obtained strain was named as an MG1655pflBgene-deleted strain (hereinafter may be simply referred to as ΔpflBstrain).

Example 18 Preparation of Escherichia coli MG1655pflB&glcDEFGene-Deleted Strain

The plasmid pTHΔglcDEF obtained in Example 16 was transformed to a ΔpflBstrain to finally give an MG1655pflB&glcDEF gene-deleted strain(hereinafter may be simply referred to as a ΔpflBΔglcDEF strain). Thedetailed method was the same as that described in Example 16.

Example 19 Construction of ΔglcDEF/pGAPfucO Strain, ΔpflB/pGAPfucOStrain and ΔpflBΔglcDEF/pGAPfucO Strain

The plasmid pGAPfucO obtained in Example 1 was transformed to theΔglcDEF strain obtained in Example 16, ΔpflB strain obtained in Example17 and ΔpflBΔglcDEF strain obtained in Example 18 respectively, andcultured on an LB agar plate containing 50 μg/mL of ampicillin at 37degree centigrade overnight to give a ΔglcDEF/pGAPfucO strain, aΔpflB/pGAPfucO strain and a ΔpflBΔglcDEF/pGAPfucO strain.

Example 20 Production of glycolic acid by ΔglcDEF/pGAPfucO strain,ΔpflB/pGAPfucO strain and ΔpflBΔglcDEF/pGAPfucO strain

The MG1655/pGAPfucO strain obtained in Example 3, and ΔglcDEF/pGAPfucOstrain, ΔpflB/pGAPfucO strain and ΔpflBΔglcDEF/pGAPfucO strain obtainedin Example 19 were cultured in the same manner as in Example 3. Themicrobial mass at 24 hours after starting the culture was collected bycentrifugation (8,000 rpm for 20 minutes) and washed with distilledwater. 75 g of the microbial mass after washing was weighed andsuspended with distilled water along with 150 g of ethylene glycol tohave 1,500 ml of final liquid amount. The suspension was transferred toa culture apparatus of DPC-2 fermentor manufactured by ABLE Corporationto carry out the reaction for 22 hours. The reaction was carried outunder the conditions including atmospheric pressure, an aeration rate of0.7 vvm, a stirring speed of 800 rpm, a reaction temperature of 35degree centigrade and pH 7.2 (adjusted with an aqueous NH₃ solution).The amount of glycolic acid, oxalic acid and acetic acid accumulated inthe obtained reaction solution, and the residual amount of ethyleneglycol were measured by HPLC according to an established method. Theresults are shown in Table 5. A by-product organic acid such as oxalicacid and acetic acid can be reduced by deletion of glcDEF gene and/orpflB gene, the conversion rate of ethylene glycol was improved, and theproductivity of glycolic acid was dramatically improved.

TABLE 5 ^(Δ)pflB MG1655/ ^(Δ)glcDEF/ ^(Δ)pflB/ ^(Δ)glcDEF/ pGAPfucOpGAPfucO pGAPfucO pGAPfucO strain strain strain strain Glycolic 102.0180.2 176.7 179.4 acid (g) Ethylene 60.5 Not 0.5 Not glycol (g) detecteddetected Oxalic acid (g) 4.06 1.11 2.88 1.23 Acetic acid (g) 0.13 0.180.10 0.10

The invention claimed is:
 1. A process for producing hydroxycarboxylicacids with aliphatic polyhydric alcohols having a terminal hydroxylgroup as a substrate, which comprises: reacting an aliphatic polyhydricalcohol having a terminal hydroxyl group with an Escherichia coli whichhas imparted or enhanced lactaldehyde reductase activity, therebyproducing a hydroxycarboxylic acid, wherein: said Escherichia colicomprises a plasmid comprising a gene encoding a lactaldehyde reductase,said aliphatic polyhydric alcohol having a terminal hydroxyl group isethylene glycol, diethylene glycol, or glycerol, wherein an endogenousglycolate oxidase and/or an endogenous pyruvate formate-lyase activityof said Escherichia coli is inactivated or decreased, and furtherwherein the yield of said hydroxycarboxylic acid is not less than 95%and the production rate is not less than 2 g/L/hr.
 2. The process as setforth in claim 1, wherein said plasmid further comprises a gene encodinga lactaldehyde dehydrogenase.
 3. The process as set forth in claim 1,wherein the hydroxycarboxylic acid is a hydroxycarboxylic acid havingoptical activity.
 4. The process as set forth in claim 2, wherein thehydroxycarboxylic acid is a hydroxycarboxylic acid having opticalactivity.
 5. A process for producing glycolic acid obtained by usingethylene glycol, the method comprising: reacting ethylene glycol with anEscherichia coli which has imparted or enhanced lactaldehyde reductaseactivity, thereby producing glycolic acid, wherein: said Escherichiacoli comprises a plasmid comprising a gene encoding lactaldehydereductase and wherein an endogenous glycolate oxidase and/or anendogenous pyruvate formate-lyase activity of said Escherichia coli isinactivated or decreased, said gene encoding lactaldehyde reductase isfunctionally linked to a promoter of a glyceraldehyde 3-phosphatedehydrogenase gene, and further wherein the yield of glycolic acid isnot less than 95% and the production rate is not less than 2 g/L/hr. 6.The process as set forth in claim 1, wherein said aliphatic polyhydricalcohol having a terminal hydroxyl group is ethylene glycol, and saidhydroxycarboxylic acid is glycolic acid.
 7. The process as set forth inclaim 2, wherein said aliphatic polyhydric alcohol having a terminalhydroxyl group is ethylene glycol, and said hydroxycarboxylic acid isglycolic acid.
 8. The process as set forth in claim 1, wherein said geneencoding said lactaldehyde reductase is functionally linked to apromoter of a glyceraldehyde 3-phosphate dehydrogenase gene.
 9. Theprocess as set forth in claim 2, wherein said gene encoding saidlactaldehyde dehydrogenase and said gene encoding said lactaldehydereductase are functionally linked to a promoter of a glyceraldehyde3-phosphate dehydrogenase gene.
 10. The process as set forth in claim 1,wherein said aliphatic polyhydric alcohol having a terminal hydroxygroup is ethylene glycol.